Fluorescence correlation spectroscopy diffusion coefficient

Fluorescence correlation spectroscopy (FCS) measures rates of diffusion, chemical reaction, and other dynamic processes of fluorescent molecules. These rates are deduced from measurements of fluorescence fluctuations that arise as molecules with specific fluorescence properties enter or leave an open sample volume by diffusion, by undergoing a chemical reaction, or by other transport or reaction processes. Studies of unfolded proteins benefit from the fact that FCS can provide information about rates of protein conformational change both by a direct readout from conformation-dependent fluorescence changes and by changes in diffusion coefficient. 

Lead, J. R., Wilkinson, K. J., Starchev, K., Canonica, S. and Buffle, J. (2000). Diffusion coefficients of humic substances as determined by fluorescence correlation spectroscopy role of solution conditions, Environ. Sci. Technol., 34, 1365-1369. 

In fluorescence correlation spectroscopy (FCS), the temporal fluctuations of the fluorescence intensity are recorded and analyzed in order to determine physical or chemical parameters such as translational diffusion coefficients, flow rates, chemical kinetic rate constants, rotational diffusion coefficients, molecular weights and aggregation. The principles of FCS for the determination of translational and rotational diffusion and chemical reactions were first described in the early 1970s. But it is only in the early 1990s that progress in instrumentation (confocal excitation, photon detection and correlation) generated renewed interest in FCS. 

Fluorescence Correlation Spectroscopy and Fluorescence Burst Analysis. Several nanoscopic chemical imaging approaches work very well for measurements of chemical kinetics, interactions, and mobility in solution. Fluorescence correlation spectroscopy (FCS) measures the temporal fluctuations of fluorescent markers as molecules diffuse or flow in solution through a femtoliter focal volume.54 Their average diffusive dwell times reveal their diffusion coefficients, and additional faster fluctuations can reveal chemical reactions and their kinetics if the reaction provides fluorescence modulation. Cross-correlation of the fluorescence of two distinguishable fluorophore types can very effectively reveal chemical binding kinetics and equilibria at nanomolar concentrations. 

In fluorescence correlation spectroscopy (FCS) a small volume element or a small area) of a sample is illuminated by a laser beam and the autocorrelation function of fluctuations in the fluorescence is determined by photon counting. From this autocorrelation function the mean number densities of the fluorophores and their diffusion coefficients can be extracted. Measurement and analysis of higher order correlation functions of the fluorescence has been shown to yield information concerning aggregation states of fluorophores ). 

Figure 33.6 Illustration of confocal volume in fluorescence correlation spectroscopy (FCS) describing the experimental principle for evaluation of diffusion coefficients from the fluctuation of photon signals, (a) Fluctuation due to large and less mobile molecules is slow and...

New fluorescence correlation spectroscopy (FCS) suitable for the observation of anomalous diffusion in polymer solution time and space dependences of diffusion coefficients. 

Fluorescence correlation spectroscopy analyses the temporal fluctuations of the fluorescence intensity by means of an autocorrelation function from which translational and rotational diffusion coefficients, flow rates and rate constants of chemical processes of single molecules can be determined. For example, the dynamics of complex formation between /3-cyclodextrin as a host for guest molecules was investigated with singlemolecule sensitivity, which revealed that the formation of an encounter complex is followed by a unimolecular inclusion reaction as the rate-limiting step.263... 

Lead JR, Wilkinson KJ, Balnois E, Cutak BJ, Larive CK, Assemi S, Beckett R Diffusion Coefficients and Polydispersities of the Suwannee River Fulvic Acid Comparison of Fluorescence Correlation Spectroscopy, Pulsed-Field Gradient Nuclear Magnetic Resonance, and Flow Field-Flow Fractionation. Environ Sci Technol 2000, 34 3508-3513. 

The amount of information contained within these bursts is large. Techniques such as fluorescence correlation spectroscopy (FCS, Chapter 2) are able to extract the average width (the amount of time) that fluctuations in the signal last for and so can measure the width of the transients (i.e. diffusion coefficients) or the width... 

Lead, J. R., K. J. Wilkinson, E. Balnois, B. J. Cutak, C. K. Larive, S. Assemi, and R. Beckett. 2000. Diffusion coefficients and polydispersities of the Suwannee River fulvic acid comparison of fluorescence correlation spectroscopy, pulsed-field gradient nuclear magnetic resonance, and flow field-flow fractionation. Environmental Science and Technology 34, no. 16 3508-3513. 

Already in 1972, Magde, Webb, and Elson published the first paper on fluorescence correlation spectroscopy yielding chemical rate constants and diffusion coefficients [2], followed by a series of further reports on these novel techniques [3-5]. However, several further developments were necessary for FCS to reach its current power which has been reviewed several times as for example in [6-10]. One key step in the evolution of FCS was its combination with confocal microscopy to enhance spatial resolution basically down to the diffraction limit of the fluorescence light and the concomitant increase in sensitivity [11]. Further important technical improvements concern the quality of optical components and the sensitivity and time resolution of detectors. Additionally, better labels and labeling strategies have become available, a point which should not be underestimated. 

Fluorescence correlation spectroscopy thus provides a way to study processes that change the translational diffusion coefficient, such as binding of a small, fluorescent ligand to a macromolecule. However, the spatial dependence of the light intensity in the focal region can be more complex than Eq. (5.83) assumes and this can add spurious components to the autocorrelation function [263]. 

At an early stage, it became apparent( 15-17) that interactions between diffusing macromolecules substantially modify what was then termed the translational diffusion constant. As had initially not been entirely transparent to the QELSS community, though it had been known elsewhere, there are in fact two translational diffusion coefficients, and (9,10). QELSS measures Dm, with complications at large q(9,10,16). If one can optically tag a few macromolecules, QELSS can also (as originally predicted(ll) and more recently confirmed(12) experimentally for fluorescence correlation spectroscopy) determine D. ... 

The additional advantage of CARS-CS over DLS and FCS is the spectral selectivity for individual chemical components in their native state, where fluorescent labeling is not desired. This may not only allow mapping of 3D diffusion coefficients, for example inside life cells, but also offer a method to monitor the specific interaction of individual components within complex systems, e.g., aggregation processes of different chemical species. Another prospect is the implementation of CARS cross-correlation spectroscopy that may allow the investigation of correlated fluctuations between two different species. These could be two distinct Raman spectral features of one and the same compound, or a specific intrinsic Raman band and an emission of a more sensitive fluorescence label [160]. 

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